1. Field of the Invention
The present invention relates generally to the detection of a target nucleic acid sequence and specifically to the detection of microsatellite DNA sequence mutations associated with a cell proliferative disorder.
2. Description of Related Art
Cancer remains a major cause of mortality worldwide. Despite advancements in diagnosis and treatment, the overall survival rate has not imrved significantly in the past twenty years. There remains an unfulfilled need for a more sensitive means of early diagnosis of tumors, before the cancer progresses.
One of the most serious cancers is bladder cancer. Bladder cancer is the fourth most common cancer in men and the eighth most common in women. Transitional cell carcinoma (TCC) of the bladder is the most common urothelial malignancy of the urinary tract, with an incidence of approximately 51,000 each year in the United States alone.
One reason that bladder cancer is so serious is because, presently, detecting and treating bladder cancer is difficult. Seventy percent of patients with an initial diagnosis of trrnsitional cell carcinoma have superficial tumors, which can be treated by transurethral resection alone. Approximately 70% of these patients continue to suffer from recurrent disease, and 15% develop lesions that invade muscle within the first two years.
Detecting tumor recurrence in patients with transitional cell carcinoma of the bladder requires close surveillance. Urine cytology is a common non-invasive procedure for the diagnosis of this disease, but it can miss up to 50% of tumors. The "gold standard" for diagnosis is cystoscopy, which allows visualization and direct biopsies of suspicious bladder lesions in the mucosa. However, because cystoscopy is an expensive and invasive procedure, it cannot be used as a general screening tool for the detection of bladder cancer.
Other serious cancers are the head and neck cancers. Head and neck cancer remains a morbid and often fatal disease. Large tumor bulk and tumor extension are predictors of a local regional recurrence and poor outcome. Detection of occult neoplastic cells in surrounding surgical margins is a strong predictor of local regional recurrence resulting in a significant decrease in overall survival
DNA contains unique sequences interspersed with moderately and highly repetitive DNA sequences. Variations in the repetitive sequence elements such as minisatellite (or variable number tandem repeat) DNA sequences and microsatellite (or variable simple sequence repeat) DNA sequences have been useful for chromosomal identification, primary gene mapping, and linkage analysis. Microsatellite DNA sequences are an especially common and highly polymorphic class of genomic elements in the human genome. One advantage to the use of repetitive sequence variations is the greater number of alleles present in populations compared with unique genetic sequence variations. Another advantage is the ability to readily detect sequence length variations using the polymerase chain reaction for the rapid and inexpensive analysis of many DNA samples.
Tumors progress through a series of genetic mutations. These genetic mutations can be used as specific markers for the detection of cancer. One set of genetic mutations that can be used to detect the presence of cancer is the loss of chromosomes. Diploid organisms, including humans, have pair of chromosomes for each member of the chromosomal set. Tumor cells will characteristically lose chromosomes, resulting in a single chromosome, rather than a pair of chromosomes, for each member of the chromosomal set. Chromosomal deletions and additions are an integral part of neoplastic progression and have been described in most kinds of cancers. A pair of chromosomes which has two alleles for a genetic locus is heterozygous for that locus; therefore, heterozygosity correlates with a cell having a pair of chromosomes. For years, these chromosomal deletions or amplifications were detected through the loss of heterozygosity.
Another of the genetic mutations used to detect the presence of cancer is genetic instability. Genetic recombination tends to occur most frequently at regions of the chromosome where the DNA is homologous (where the DNA has a high degree of sequence similarity). Where a DNA sequence is repetitive, the DNA homology is greater. The DNA homology occurs not only at the same genetic locus on the other pair of chromosomes, but also on other genetic loci or within the same locus on the same chromosome. Normal (non-tumor) cells tend to suppress this genetic recombination. Tumor cells, however, characteristically undergo increased genetic recombination. Where a DNA sequence is repetitive, genetic recombination can result in the loss of repeat DNA sequences or the gain of repeat DNA sequences at a genetic locus.
Microsatellite DNA instability has been described in human cancers. Microsatellite DNA instability is an important feature of tumors from hereditary non-polyposis colorectal carcinoma patients Peltomaki et al., Science, 260: 810 (1993); Aaltonen et al., Science, 260: 812 (1993); Thibodeau et al., Science, 260: 816 (1993)). Microsatellite DNA instability by expansion or deletion of repeat elements has also been reported in colorectal, endometrial, breast, gastric, pancreatic, and bladder neoplastic tissues (Risinger et al., Cancer Res., 53: 5100 (1993); Had et al., Cancer Res., 53: 5087 (1993); Peltomaki et al., Cancer Res., 53: 5853 (1993); Gonzalez-Zulueta et al., Cancer Res., 53: 5620 (1993)).
Some methods have been developed to detect the multiple genetic changes that occur during the development of primary bladder cancer. For example, mutations in the tumor suppressor gene p53 signal the progression to invasiveness and have been successfully used as molecular markers to detect cancer cells in urine samples. However, this diagnostic strategy has limited clinical application because the techniques are cumbersome and because p53 mutations appear relatively late in the disease.
Because early diagnosis of bladder cancer is critical for successful treatment, there is a pressing need for more sensitive and cost-effective diagnostic tools. Both patients and physicians would benefit from the development of improved non-invasive methods for cancer surveillance.